Death due to prostate cancer (PCa) generally results when patients develop metastatic castration-resistant prostate cancer (mCRPC). While current treatments for mCRPC improve survival, the disease still remains incurable, and treatments result in severe side effects, such as impotence and incontinence. Current methods to detect PCa and monitor treatment outcomes are typically invasive, indicating a need for new imaging agents that use sensitive molecular imaging technologies such as PET (positron emission tomography). Therapeutic peptides, with cancer cell specific activity, are an especially promising treatment option for mCRPC. Recently, we discovered CT20p, a novel mitotoxic peptide that targets cancer-specific differences in mitochondrial physiology. CT20p is a promising anti-metastatic agent because it causes detachment-induced cell death; however, to develop the clinical use of CT20p for mCRPC, there are challenges that need to be met, such as low stability in serum. New platform technologies for the delivery and monitoring of therapeutic peptides to areas of disease are urgently needed. Our objective is to develop a targeted molecular nanotheranostic (dual therapy and diagnostic) platform that delivers CT20p in high concentrations to PCa and has the capacity for imaging peptide efficacy in murine models of PCa. To deliver CT20p to PCa, the peptide will be encapsulated within hyperbranched polyester nanoparticles (HBPE-NPs) that are functionalized with polyglutamated folates, that natural ligand for a PCa-specific cell surface protein, PSMA. PSMA is highly expressed in PCa tumors and metastatic lesions but not normal prostate. To endow our NPs with imaging capabilities, the polymer will be modified to graft desferrioxamine (DFO), a chelating ligand for stable encapsulation of a89Zr-PET imaging probe. We hypothesize that PSMA-targeted HBPE[CT20p]NPs, co-encapsulated with 89Zr, will yield a powerful therapeutic platform to reduce PCa growth and metastatic spread, while enabling assessment of particle bio- distribution in the proposed pre-clinical studies. Three aims are planned in which the synthesis of HBPE- DFO[CT20p]-NPs will be optimized to obtain effective chelation of 89Zr, pegylation and CT20p loading (Aim 1), and a series of polyglutamated folate-HBPE-DFO[CT20p]-NPs will be synthesized and tested to target PCa cells via PSMA (Aim 2). PET imaging, using murine models of PCa, will be used to assess delivery and efficacy of CT20p and pharmacokinetics studies will be performed. The potential clinical value of our HBPE- DFO[CT20p]-NPs will be investigated in murine models of PCa, using mice that are intact or castrated, and in models of lymph node and bone metastasis (Aim 3). The outcome of our research will be PSMA-targeted, HBPE-DFO[CT20p]-NPs (without 89Zr) that can be directly used for the treatment of mCRPC without the side effects associated with current therapies, while the theranostic version (with 89Zr) will provide the pre-clinical data to advance the use of PET imaging for monitoring fast growing prostate tumors and treatment outcomes.